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INFORMATION ON MASTER’THESIS
1. Full name: Tran Thi Tinh
2. Sex: Female
3. Date of birth: 15/10/1982
4. Place of birth: Tuyen Quang
5. Admission decision number:
Dated
6. Changes in academic process: No
7. Official thesis title: “The study of rapid detection of avian influenza virus (A/H5N1)
in clinical samples by multiplex reverse transcription polymerase chain reaction”.
(This study were carried out in the laboratory of Thai Binh Medical University)
8. Major:
Experimental Biology
9. Code: 60 42 30
10. Supervisors: Prof. Luong Xuan Hien - Thai Binh Medical University
11. Summary of the finding of the thesis:
I. Introduction:
Avian influenza (AI) is a highly contagious disease cause by type A influenza
virus, a member of the Orthomyxoviridae family. Influenza A virus causes major and
frequently fatal in birds, as well as in mammals including humans. At times of influenza
A virus subtype H5N1 outbreaks, control of further outbreaks and re-emergence
prevention are very important. Therefore, rapid accurate and sensitive molecular
diagnostic methods is crucical for large-scale screening of this virus. So that, we study
objective with aim:
1. Design and select appropriate primers for detection of avian influenza virus by
multiplex reverse transcription polymerase chain reaction.
2. Optimize conditions for multiplex RT-PCR: appropriated time and temperature
for primer annealing, concentration of participated components,..
3. Test some collected clinical samples with multiplex RT-PCR for detection of
avian influenza virus.
II. Materials and methods
Source of Viruses: RNA
of
influenza
A
virus
subtype
H5N1
obtained from The Institute of Biotechnology(IBT). Positive
samples
were
(N=14)
collected
for
from
H5N1
subtype
diseased
of
avian
influenza
and
A
viruses
human;
samples
(N=30)
were
collected
from
diseased
avian
in
influenza
pandemic which outbreak in 2009.
RNA extraction: RNA extraction was performed using the QIAmp Viral RNA mini Kit
according to the manufacturer’s specifications.
Primer Designs: Nucleotide sequences of the matrix (M), hemagglutinin (HA) and
neuraminidase (NA) genes were taken from the Influenza Virus Resource of the NCBI
database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). These sequences were
aligned using the ClustalX 2.0.11 program. The primers were designed and analyzed by
using the PCR primer software: BioEid, FastPCR 5.4 to ascertain that they could be
combined in a mutiplex RT-PCR format.
Mutiplex RT-PCR optimality: The multiplex RT-PCR reaction was performed
with the Qiagen OneStep RT-PCR Kit according to the manufacturer’s specifications in a
Master cycler C1000 (Biorad). The multiplex RT-PCR were tested and optimized in
different conditions on primer concentrations, annealing temperature…
After PCR
amplification, 10 µl of PCR products were subjected to 2% agarose gel electrophoresis.
Gel stained with 0.5 µg/ml ethidium bromide solution for 15 min and visualized on a
UVP. The expected sizes of each PCR product are compare to standar DNA maker.
Specificity test of mutiplex RT-PCR: For positive specificity test, RNA extracted
from specimens positive for influenza A virus subtype H1N1, subtype H3, influenza B
virus (National Institute of Hygiene and Epidemiology provides), influenza A virus
subtype H5 (National centre for veterinary diagnostics provides) and DNA, RNA
samples being in our lab were amplified by the mutiplex RT-PCR to check cross
reaction occurred against other specimens.
Sensitivity test of mutiplex RT-PCR: The genes of influenza A virus subtype H5N1
were used to construct plasmid DNAs by insertion of the M, HA and NA genes in to the
pJET1.2/blunt Vector (Fermentas) by CloneJETTM PCR Cloning Kit #K1231,
#K1232.
In
vitro
transcription
was
performed
by
using
TranscriptAid™ T7 High Yield Transcription Kit (Fermentas) following the manufacturer’s
recommendations. The concentration of the transcribed RNAs was calculated by measuring
absorbance at 260 nm. The RNAs were then serially diluted 10-fold, ranging from stock
concentration to 10 copies/ µl to perform sensitivity tests.
III. Result
Primer Designs: After analysis sequences of M, HA, NA gen in influenenza A
virus subtype H5N1 optained from GenBank by design softwares, three primer pairs were
chose to perform mutiplex RT-PCR for detecting influenza A virus subtype:
Primers
Sequences (5’-3’)
Position
Tm
DiagMF
GTCTTCTAACCGAGGTCGAAAC
5-26
55.1
DiagMR
GTGACAGGATTGGTCTTGTCTT
158-139
55.8
DiagH5F
AGTGATCAGATTTGCATTGGTTAC
46-69
54.6
DiagH5R
GACCAAGAACTTTTGGGGATG
416-396
55.4
DiagN1F
CCAGTTGGTTGACAATTGGAAT
503-524
54.5
DiagN1R
GCATCAGGATAACAGGAGCA
794-775
55.4
Size
154 bp
371 bp
292 bp
Mutiplex RT-PCR: RT-PCR reaction to each grade separately primer were
conducted to determine the conditions (primer concentration, temperature for primer
annealing, extensible time, the number of cycles) the most appropriate for each primer.
Then, RT-PCR reaction tested with all of 3 primer sets to determine the optimal
conditions for reaction. The results of the test showed that mutiplex RT-PCR for the best
result in the primer concentration for the M, HA and NA gene respectively 0.3 µM, 0.5
µM and 0.4 µM; reaction is performed with thermal program: reverse transcription at
50°C for 30 min; initial denaturation at 95oC for 15 minutes followed by 45 cycles
(94oC: 30 seconds; 57oC: 30 seconds; 72oC: 30 seconds) ; 72oC in 5 minutes.
Specificity test of mutiplex RT-PCR: The mutiplex RT-PCR were performed
with RNA of some specimens (influenza virus and RNA, DNA sample in Lab). As a
result, rection only amplify genes of influenza A virus subtype H5N1 (M, H5 and N1).
Specificity test of mutiplex RT-PCR:
The multiplex RT-PCR were performed with standard samples having different
concentration from 10 to 109 copies/µl and 10 µl of PCR product subjected to 2%
agarose gel electrophoresis. Amplified products visible at RNA concentrations as low as
101 copies/ µl
Applying the technique of mutiplex RT-PCR detects influenza A/H5N1 in the
clinical samples:
Mutiplex RT-PCR technique tested on 14 positive samples for A/H5N1 influenza
virus. As a result, mutiplex RT-PCR reactions were detected A/H5N1 influenza virus in
all 14 samples.
Mutiplex RT-PCR technique identified H5N1 influenza in 30 samples (collected
from infected poultry during outbreaks of influenza). Findings are 9 samples positive for
H5N1 influenza (30%) and 21 negative samples for H5N1 influenza (70%).
Conclusion:
- Designed to be three pairs of gene-specific primers with M, HA and NA gene
used to detect avian influenza A virus H5N1 in clinical specimens by RT-PCR
reactions.
- Most successful construction mutiplex RT-PCR method allows rapid detection of
influenza A virus H5N1 with high sensitivity and specificity and can be applied for
detecting infection with influenza A virus H5N1 in avian and human.
- Mutiplex RT-PCR method only specific to the influenza A virus H5N1, no crossreactivity with other species. Technique able to detect in the sample having 10 or more
copies of virus.
12. Practical applicability: Applying the technique of mutiplex RT-PCR detects
influenza A virus H5N1 in clinical samples in avian and human.
Date: 2/12/2011
Signature:
Full name:Tran Thi Tinh